The present invention relates to a novel primary anticoagulant for blood and its use in the preparation of platelets stored prior to transfusion into a patient. Platelets are obtained as a by product from whole blood donations and from plateletphersis procedures. Typically they are now stored at 22.+-.2.degree. C. in their own plasma within a plastic container whose walls are permeable to atmospheric gases. The plasma associated with these platelets normally contains all the ingredients of normal plasma plus ingredients in the primary anticoagulant which result in a dextrose concentration five times the physiologic concentration. The dextrose is added to the primary anticoagulant for the benefit of red cells which require it during storage, and dextrose is generally accepted to be required for platelet storage as well.
In routine blood banking practice, the primary anticoagulant which is utilized in citrate-phosphate-dextrose (CPD). CPD is composed of:
______________________________________ trisodium citrate(dihydrate) 26.30 g citric acid (monohydrate) 3.27 g sodium dihydrogen phosphate 2.22 g (monohydrate) dextrose 25.50 g water to 1 liter ______________________________________
From Mollison, P. L., Blood Transfusion in Clinical Medicine, 7th Edition, Blackwell, 1983.
It is to be noted that the final concentration of the components of the anticoagulant in the platelet concentrate derived from the blood-anticoagulant mixture may be expressed as a millimolar (mM) concentration or as milliequivalents (meg) per liter. By convention, the final concentration of "citrate" has often been expressed as mM by adding the millimolar concentration of trisodium citrate and citric acid. Since citrate is an anion when in solution, the concentration when derived from several sources may more properly be expressed as meq per liter. For solutions of trisodium citrate and citric acid, the relationship between the two modes of expressing concentration is 3 equivalents per mole. Such an anticoagulant mixture of trisodium citrate and citric acid is referred to herein as acid citrate.
Donations of a unit of blood (63 ml of CPD mixed with 450 ml of whole blood) are processed by centrifugation into three fractions: red cells, plasma, and platelets. The volume of packed red cells from a unit of blood is approximately 180 ml with a remaining volume of plasma and anticoagulant of about 333 ml. As used in the remainder of this application, the term plasma includes any anticoagulant which has been added thereto at the time of blood collection. The red cells are typically suspended in approximately 45 ml of plasma. Platelets are suspended in approximately 50 ml of plasma. This platelet containing product is typically referred to as a platelet concentrate. The remaining 238 ml of plasma is frozen as fresh plasma.
A great deal is known about human platelt cells. General papers describing techniques, materials and methods for storage of platelets are described by Murphy et al. in "Improved Storage of Platelets for Transfusion in a New Container", Blood 60(1): 194-200 (July, 1982); by Murphy in "The Preparation and Storage of Platelets for Transfusion", Mammon, Barnhart, Lusher and Walsh, PJD Publications, Ltd., Westbury, N.Y. (1980); by Murphy in "Platelet Transfusion", Progress in Hemostasis and Thrombosis, Vol. III, Edited by Theodore Spaet, Grune and Stratton, Inc. (1976); by Murphy et al. in "Platelet Storage at 22.degree. C.: Role of Gas Transport Across Plastic Containers in Maintenance of Viability", Blood 46(2): 209-218 (1975); by Kilkson, Holme and Murphy in "Platelet Metabolism During Storage of Platelet Concentrates at 22.degree. C.", Blood 64(2): 406-414 (1984); by Murphy in "Platelet Storage for Transfusion", Seminars in Hematology 22(3): 165-177 ( 1985); by Simon, Nelson, Carmen and Murphy in "Extension of Platelet Concentrate Storage", Transfusion 23: 207-212 (1983); by Cesar, Diminno, Alam, Silver and Murphy in "Plasma Free Fatty Acid Metabolism During Storage of Platelet Concentrates for Transfusion", Transfusion 27(5): 434-437 (1987); each of which publications is hereby incorporated by reference as if more fully set forth herein.
There exists a considerable body of prior art concerning storage of platelets. Prior work has shown that the duration of platelet storage is limited by the continuing production of lactic acid from dextrose by the platelets. Although this provides energy for the platelets, the lactic acid acidifies the medium, which acidity eventually destroys the platelets. It has also been shown that platelets consume oxygen during storage for energy production, the end product of which process is a gas, CO.sub.2 which, unlike lactic acid, can leave the platelet concentrate through the plastic walls of the container in which it is normally stored. The production of CO.sub.2 does not acidify the storage medium for the platelets. In addition to the glycolysis of dextrose, fatty acids and amino acids typically present in the plasma may be used as substrates for oxidative metabolism of stored platelet cells.
However, most platelet storage media contain glucose. In U.S. Pat. No. 4,695,460 (Holme), a synthetic platelet storage media is disclosed containing 3.0-7.5 grams of dextrose, 3.0-6.0 grams of sodium citrate, and 2.0-4.2 grams of sodium bicarbonate. U.S. Pat. No. 4,447,415 (Rock) discloses a number of storage solutions, all but one containing glucose. The one non-glucose containing solution contains trypsin which would be deleterious to the survival of platelets. It has been appreciated that platelet storage in a medium essentially free of glucose could be advantageous. For example, in U.S. Pat. No. 4,828,976, Murphy discloses a glucose free media for storing blood platelets. To store platelets for periods in excess of 5 days, it is taught that the storage media should be essentially free of glucose. It is also disclosed that it is the presence of glucose that leads to the generation of lactic acid which adversely affects platelet viability. However, Murphy utilized the conventional glucose-containing primary anticoagulant and omitted glucose only from the storage media.
The anticoagulants currently used for blood donations are designed to optimize the subsequent storage of red blood cells. The acidity of the citrate-citric acid mixture yields pH, approximately 7.1, in the anticoagulated blood. Higher pHs speed red blood cell glycolysis and lower pHs speed 2,3 DPG disappearance. The final citrate concentration, however, is 40% higher than that actually required to anticoagulate the blood. Glucose is added so that its final concentration in the anticoagulated blood is 25 mM thus providing substrate for red cell glycolysis.
Many of these conditions which have been conventionally employed may not be optimal for the preparation and storage of platelet concentrates (PC) Since red cell additive solutions are coming into widespread use, new primary anticoagulants should be designed to optimize platelet transfusion therapy.
Notwithstanding the considerable work conducted in this area, a need still exists for means to improve the storage of platelets in a viable condition.